The protamine P1 genes from two monotremes, platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus) were isolated after polymerase-chain-reaction amplification then cloned and sequenced. The two protamine P1 genes are of 290 bp and 311 bp for platypus and echidna, respectively, and are clearly orthologous to the published sequences of protamine P1 genes of eutherian mammals and birds. Both genes contain an intron, like the mammals and marsupials and unlike the bird P1 genes that are intronless. The deduced protein sequences from the coding areas of the platypus and echidna protamine P1 genes do not contain any cysteine residues. This absence of cysteine residues leaves the sperm nuclei susceptible to disruption in vitro by exposure to increasing ionic strength and is a characteristic of fish, birds and marsupials. In contrast, the P1 protamines of placental mammals invariably contain 6-9 cysteine residues that, as a result of the formation of intermolecular and intramolecular disulfide bridges, significantly increase the stability of the sperm nuclei that can only be disrupted following disulfide-bond cleavage. Phylogenetic analysis of the protamine P1 gene sequences indicates that the monotremes occupy a position half-way between the eutherian mammals and birds. From the DNA sequences we estimate the time of divergence of the platypus and the echidna to be around 22 million years ago. This date agrees very well with the published estimates of divergence based on other criteria.

We present DNA-hybridization data on 21 amniotes and two anurans showing that discrimination is obtained among most of these at the class and lower levels. Trees generated from these data largely agree with conventional views, for example in not associating birds and mammals. However, the sister relationships found here of the monotremes to marsupials, and of turtles to the alligator, are surprising results which are nonetheless consistent with the results of some other studies. The Marsupionta hypothesis of Gregory is reviewed, as are opinions about the placement of chelonians. Anatomical and reproductive data considered by Gregory do not unequivocally preclude a marsupial-monotreme special relationship, and there is other recent evidence for placing turtles within the Diapsida. We conclude that the evidential meaning of the molecular data is as shown in the trees, but that the topologies may be influenced by a base-compositional bias producing a seemingly slow evolutionary rate in monotremes, or by algorithmic artefacts (in the case of turtles as well).

A reconstruction of the skull, dentary and dentition of the middle Miocene ornithorhynchid Obdurodon dicksoni has been made possible by acquisition of nearly complete cranial and dental material. Access to new anatomical work on the living platypus, Ornithorhynchus anatinus, and the present comparative study of the cranial foramina of Ob. dicksoni and Or. anatinus have provided new insights into the evolution of the ornithorhynchid skull. The hypertrophied bill in Ob. dicksoni is seen here as possibly apomorphic, although evidence from ontogenetic studies of Or. anatinus suggests that the basic form of the bill in Ob. dicksoni (where the rostral crura meet at the midline) may be ancestral to the form of the bill in Or. anatinus (where the rostral crura meet at the midline in the embryonic platypus but diverge in the adult). Differences in the relative positions of cranial structures, and in the relationships of certain cranial foramina, indicate that the cranium may have become secondarily shortened in Or. anatinus, possibly evolving from a more elongate skull type such as that of Ob. dicksoni. The plesiomorphic dentary of Ob. dicksoni, with well-developed coronoid and angular processes, contrasts with the dentary of Or. anatinus, in which the processes are almost vestigial, as well as with the dentary of the late Oligocene, congeneric Ob. insignis, in which the angular process appears to be reduced (the coronoid process is missing). In this regard the dentary of Ob. insignis seems to be morphologically closer to Or. anatinus than is the dentary of the younger Ob. dicksoni. Phylogenetic conclusions differ from previous analyses in viewing the northern Australian Ob. dicksoni as possibly derived in possessing a hypertrophied bill and dorsoventrally flattened skull and dentary, perhaps being a specialized branch of the Obdurodon line rather than ancestral to species of Ornithorhynchus. The presence of functional teeth and the robust, flattened skull and dentary in Ob. dicksoni argue for differences in diet and lifestyle between this extinct ornithorhynchid and the living Ornithorhynchus.

An outstanding problem in mammal phylogeny is the relationship of the aardvark (Orycteropus afer), the only living species of the order Tubulidentata, to the extant eutherian lineages. In order to examine this problem the complete mitochondrial DNA (mtDNA) molecule of the aardvark was sequenced and analysed. The aardvark tRNA-Ser (UCN) differs from that of other mammalian mtDNAs reported and appears to have reversed to the ancestral secondary structure of non-mammalian vertebrates and mitochondrial tRNAs in general. Phylogenetic analysis of 12 concatenated protein-coding genes (3325 amino acids) included the aardvark and 15 additional eutherians, two marsupials and a monotreme. The most strongly supported tree identified the aardvark as a sister group of a clade including the armadillo (Xenarthra) and the Cetferungulata (carnivores, perissodactyls, artiodactyls and cetaceans). By applying three molecular calibration points the divergence between the aardvark and armadillo-cetferungulates was estimated at ca. 90 million years before present.